Hop2-Mnd1 and Swi5-Sfr1 stimulate Dmc1 filament assembly using distinct mechanisms

Area of Science:

• * Molecular Biology
• * Genetics
• * Biochemistry

Background:

• * Meiosis involves homologous chromosome pairing and strand exchange mediated by Dmc1 and Rad51 recombinases.
• * Fission yeast proteins Swi5-Sfr1 and Hop2-Mnd1 are known to stimulate Dmc1-mediated recombination.
• * The precise molecular mechanisms by which Swi5-Sfr1 and Hop2-Mnd1 stimulate Dmc1 remain largely uncharacterized.

Purpose of the Study:

• * To elucidate the molecular mechanisms by which Hop2-Mnd1 and Swi5-Sfr1 stimulate Dmc1 recombinase activity.
• * To investigate the distinct roles of Hop2-Mnd1 and Swi5-Sfr1 in Dmc1 filament assembly on single-stranded DNA (ssDNA).
• * To determine how the combined action of these accessory proteins affects Dmc1 nucleation and filament formation.

Main Methods:

• * Single-molecule fluorescence resonance energy transfer (smFRET) experiments to monitor Dmc1 filament dynamics.
• * Tethered particle motion (TPM) assays to assess Dmc1 binding and assembly on DNA substrates.
• * Kinetic analyses of Dmc1 binding and dissociation rates in the presence and absence of accessory proteins.
• * Order of addition experiments to delineate the sequential roles of accessory proteins and Dmc1.

Main Results:

• * Both Hop2-Mnd1 and Swi5-Sfr1 individually enhance Dmc1 filament assembly on ssDNA.
• * Combined presence of Hop2-Mnd1 and Swi5-Sfr1 leads to further stimulation of Dmc1 filament formation.
• * Hop2-Mnd1 increases the rate of Dmc1 binding (enhances nucleation), while Swi5-Sfr1 decreases the dissociation rate of Dmc1 during nucleation.
• * Hop2-Mnd1 facilitates Dmc1 nucleation at single-stranded/double-stranded DNA junctions, with its presence shortening nucleation times.

Conclusions:

• * Hop2-Mnd1 and Swi5-Sfr1 act at distinct steps to promote Dmc1 filament assembly during meiotic recombination.
• * Hop2-Mnd1 functions in recruiting Dmc1 and promoting its nucleation at DNA junctions.
• * Swi5-Sfr1 stabilizes Dmc1 filaments by reducing dissociation during the critical nucleation phase.
• * The regulation of Dmc1 activity is dictated by the DNA-binding properties of accessory proteins and recombinase nucleation preferences.